Phage Display Panning on Silica: Optimization of Elution Conditions for Selection of Strong Binders

Phage display panning is a powerful tool to select strong peptide binders to a given target, and when applied to inorganic materials (e.g., silica) as a target, it provides information on binding events and molecular recognition at the peptide–mineral interface. The panning process has limitations with the phage chemical elution being affected by bias toward positively charged binders, resulting in the potential loss of information on binder diversity; the presence of fast growing phages with an intrinsic growth advantage; and the presence of false positives from target unrelated peptides. To overcome some of these limitations, we developed a panning approach based on the sequential use of different eluents (Gly-HCl, pH-2.2; MgCl2, pH-6.1; and TEA, pH-11.0), or pH conditions (Gly-HCl 2.2 < pH < 11.0) that allows the identification of a diverse and comprehensive pool of strong binders. We have assessed and tested the authenticity of the identified silica binders via a complementary experimental (in vivo phage recovery rates and TEM imaging) and bioinformatics approach. We provide experimental evidence of the nonspecificity of the Gly-HCl eluent as typically used. Using a fluorimetric assay, we investigate in vitro binding of two peptides that differ by pI–S4 (HYIDFRW, pI 7.80) and S5 (YSLKQYQ, pI 9.44)—modified at the C terminal with an amide group to simulate net charges in the phage display system, confirming the vital role of electrostatic interactions as driving binding forces in the phage panning process. The presented optimized phage panning approach provides an opportunity to match known surface interactions at play with suitable elution conditions; to select only sequences relevant to a particular interfacial system. The approach has the potential to open up avenues to design interfacial systems to advance our understanding of peptide-assisted mineral growth, among other possibilities.

The library manufacturer's instructios were followed for all amplification and titering procedures.Changes to the manufacturer's instructions were applied to washing, elution and neutralisation steps to obtain the three panning protocols below.
Gly-HCl eluates were neutralised with 150 µl of 1 M Tris HCl at pH-9.1, while 4 M MgCl 2 and triethylamine eluates were neutralised by adding 50 µl of 1 M Tris HCl at pH 9.1 or 150 µl of 1 M Tris HCl at pH 5, respectively prior to amplification.
For each of the three different eluents, up to 3 rounds of panning were performed before titering and plaque selection via the white-blue screening method.Selected plaques were then amplified and sequenced.

1.2
Repanning experiments: performed to cross check the reproducibility of the identified phage displayed sequences.In this process 50 µl of the amplified phage pool obtained from round 2 of separate conventional panning experiments with the three different elutents were mixed together and named 'mix of amplified phage'.This mix was then repanned against fresh silica, followed by 10 washes with TBST 0.5% -0.7% in successive rounds, eluted at low and high pH values (Gly-HCl pH 2.2 and TEA pH 11, respectively) and sequenced at rounds 3 for Gly-HCl pH 2.2; and at round 4 for TEA pH 11 (see table SI-2).
Eluted phage clones were screened after three rounds of panning following manufacturer's instructions.

Binding studies:
2.1 Peptide synthesis: Peptides were synthesized using an automated single channel microwave assisted peptide synthesizer (CEM Liberty1) following standard Fmoc chemistry and starting from preloaded Wang resins as solid supports to initiate the peptide synthesis.The amide derivatives were prepared using Fmoc-Rink Amide ProTide Resins.These synthesized peptides were characterized by HPLC and mass spectrometry.Peptide purity by HPLC was in the range 78 to 94%.

2.2
In vitro silica binding studies: suspensions of silica nanoparticles (1 mg/ ml) in phosphate-buffered saline buffer were sonicated for 1 hour, then peptide was added to achieve the desired initial peptide concentration in the range 0.2-1.6 mM.The mixtures were then shaken vigorously and left to equilibrate for 1 hour at room temperature.The silica was separated by centrifugation (12,000 rpm for 5 min) and the supernatant was analysed by fluorimetric assay to quantify the amount of peptide left in solution. 2The amount of peptide bound was then calculated by difference.

Relative binding affinity (phage binding assay):
A selection of 15 clones were individually amplified and screened against fresh silica.Silica (1mg) was washed 10 times with TBST (0.3% TWEEN [v/v] in 1 ml TBS) at pH 7.5 then incubated with 0.01 ml of 2.5E+10 pfu/ml of phage clones (input phage), and binding affinity determined via titer assay, and calculated as the ratio of output phages to input phages.Dilution factor was 10E+04 for all peptide displaying clones, and 10E+03 for wild MK13 control.

Transmission electron microscopy (TEM) imaging:
The amplified phage clones displaying each of the selected peptides YSLKQYQ and ADIRHIK were mixed with silica nanoparticles suspensions (1mg/ml) and incubated on either Poly-L-lysine and/ or paraformaldehyde coated TEM grids.The grid containing silica-phage suspension was washed three times with distilled water to remove weakly bound phage and stained with a drop (roughly 5 μl) of diluted aqueous EM stain 336 (Agar Scientific) then left for 30 min at room temperature.The grids were then air dried before washing three times with distilled water (roughly 5 μl each wash).The airdried samples were then visualised using a JEOL 2010 TEM at 200 KeV using a LaB 6 filament to confirm phage-silica binding.For comparison, silica alone, silica with the dye and a drop of the Ph.D.™-7 phage display peptide library (New England Biolabs) with dye was observed under TEM.Control data for silica + dye and phage + dye is shown in Figure S1.

Bioinformatics
Sequences were analysed by tools available on the BDB databank. 4These tools were developed using machine learning algorithms and three predictors namely PhD7Faster, SABinder and PSBinder were employed.The PhD7Faster tool was used to predict if phages bearing randomly displayed peptides from the Ph.D.-7 library might grow faster due to propagation advantage. 5The silica binders identified in this study were checked by entering the sequence data either in FASTA or raw sequence data format.The threshold to distinguish between predicted positives and negatives (tp) was set to 0.5 with a peptide being predicted to be a target unrelated peptide (TUPs) if the probability is 0.5 or higher.
EXPERIMENTAL DATA Table S3.Experimental results of optimised sequential panning approach using Gly-HCl at different pH values.In bold are indicated frequently identified silica binders.

3 Figure S1 .
Figure S1.Control TEM images of a) silica NPs (82nm) without EM stain 336; b) silica NPs with EM stain 336; c) M13 Ph.D.7 phage library stained with EM stain 336.Phage structures are indicated by white arrows.

Table S1 .
Complete list of sequences isolated from conventional panning experiments after three rounds of panning.

Table S2 .
Complete list of sequences isolated from repanning experiments showing recurrence of silica binders previously identified after three rounds of conventional panning.

Table S4 .
Phage titer calculations for relative binding assay.Experiment was run twice.Bound phage was calculated from equation SI-1.Input phage is 2.5E+05

Table S5 .
Mimo search/ scan/ Blast analysis results of silica binders identified.Frequently identified silica binders that showed hits for other target materials indicating that they might be promiscuous binders.